Subscriber access provided by Caltech Library Letter Synthesis and Biological Evaluation of Pyrroloindolines as Positive Allosteric Modulators of the #1#2#2 GABAA Receptor Annet E.M. Blom, Justin Y. Su, Lindsay M. Repka, Sarah E. Reisman, and Dennis A. Dougherty ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00340 • Publication Date (Web): 15 Sep 2020 Downloaded from pubs.acs.org on September 16, 2020

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1 2 3 4 5 6 7 Synthesis and Biological Evaluation of Pyrroloindolines as Positive 8 Allosteric Modulators of the α1β2γ2 GABAA Receptor 9 10 Annet E. M. Blom, Justin Y. Su, Lindsay M. Repka, Sarah E. Reisman*, Dennis A. Dougherty* 11 12 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United 13 States 14 15 16 ABSTRACT: γ-Aminobutyric acid type A (GABAA) receptors are key mediators of central inhibitory neurotransmission and have been 17 implicated in several disorders of the central nervous system. Some positive allosteric modulators (PAMs) of this receptor provide 18 great therapeutic benefits to patients. However, adverse effects remain a challenge. Selective targeting of GABAA receptors could 19 mitigate this problem. Here, we describe the synthesis and functional evaluation of a novel series of pyrroloindolines that display 20 significant modulation of the GABAA receptor, acting as PAMs. We found that halogen incorporation at the C5 position greatly 21 increased the PAM potency relative to the parent ligand, while substitutions at other positions generally decreased potency. Mutagenesis studies suggest that the binding site lies at the top of the transmembrane domain. 22 23 24

25 KEYWORDS: GABAA receptor, Cys-loop, positive allosteric modulator, ion channel, pyrroloindoline. 26

27 INTRODUCTION γ-Aminobutyric acid type A (GABAA) been identified and several of the positive allosteric 28 receptors are key mediators of central inhibitory modulators are widely used to treat anxiety and panic 29 neurotransmission, and as such these receptors have been disorders.9,10 30 drug targets for numerous central nervous system (CNS) Although GABAA receptor modulators have a proven 1–3 31 disorders. The GABAA receptor is an anion-selective, therapeutic benefit, adverse effects remain a problem.11,12 32 pentameric, ligand-gated ion channel that is part of the larger Additionally, elucidating functions of individual subtypes is Cys-loop receptor family. A functional receptor results from 33 crucial for a better understanding of the GABAA receptor’s role 34 the assembly of five homologous subunits. A total of 19 in health and disease. Therefore, recent efforts have focused 35 homologous subunits exist, and they assemble into at least 30 on finding subtype-selective modulators. Various novel 4 36 different functional subtypes in vivo. Some types, including modulators have been derived for the α+/β- interface.13,14 For 37 those comprised of α1β2γ2 subunits, are predominantly example, a series of pyrazolopyridinones developed by 38 expressed at the post-synaptic termini and mediate phasic Blackaby et al. showed increased selectivity for α3β3γ2 over 39 inhibition, while others are located at extrasynaptic sites and α1β3γ2.15 Two different series of pyrazoloquinolinones 4–6 40 mediate tonic inhibition. The large diversity of subtypes and exhibited selectivity for α6β3γ2 and β1-containing receptors, 41 differential localization in the brain emphasize their respectively.16,17 importance, but also present a challenge, as current GABA 42 A Physostigmine (1, Figure 1), also known as eserine or receptor therapeutics modulate a broad range of subtypes, 43 antilirium, is a reversible acetylcholinesterase inhibitor18 that which can result in adverse effects. 44 has been used to treat glaucoma and delayed gastric 45 Each GABAA subunit consists of an N-terminal extracellular emptying.19,20 In addition, it has been found to potentiate and 46 domain (ECD), a transmembrane domain (TMD) that inhibit nicotinic acetylcholine receptors, another member of 47 comprises four transmembrane α-helices (M1-M4), an the Cys-loop receptor family.21–23 48 extracellular M2-M3 loop and C-terminus, and an intracellular 7 49 domain composed predominantly of the M3-M4 loop. Receptor activation occurs upon binding of an agonist to the 50 orthosteric site, which is located in the ECD at the β+/α- 51 subunit interfaces. This activation can be modulated by 52 additional binding of other ligands to several allosteric sites on 53 the pentameric complex.8 Positive allosteric modulators 54 (PAMs) potentiate the evoked response by an agonist, while 55 negative allosteric modulators (NAMs) inhibit that response.9 56 Figure 1. Chemical structures of selected pyrroloindolines. Over the years various modulators of GABAA receptors have 57 58 59 60 ACS Paragon Plus Environment ACS Medicinal Chemistry Letters Page 2 of 9

As a result of our interest in the synthesis of pyrroloindoline In a preliminary screen, five pyrroloindoline compounds 1 natural products, we have prepared a number of new, non- including (±)-2, were tested for modulation of eight 2 natural pyrroloindoline compounds.24–26 Given their structural pentameric ligand-gated ion channels (pLGICs): muscle type

3 similarity to other modulators of Cys loop receptors, we nAChR, α4β2 nAChR, α7 nAChR, 5-HT3A receptor, α1β2γ2 4 screened a representative collection of these structures, and GABAA receptor, α1β2 GABAA receptor, GluR2, and the glycine 5 found that compounds bearing aryl substitution at C8a can act receptor. This assay identified pyrroloindoline (±)-2 as a potent 27,28 6 as PAMs of GABAA receptors. Here, we report the synthesis PAM of the α1β2γ2 GABAA receptor (Table S1 and Figure 27,28 7 of pyrroloindoline (+)-2 (Figure 1) and modification of this S1). Although no GABAA receptor activity has been 8 scaffold by substitution at N1, C3a, C5 and C8a, yielding a novel previously reported for physostigmine, compound (±)-2 9 series of GABAA receptor ligands. All of the compounds were appears to selectively potentiate α1β2γ2 GABAA receptors 10 tested for agonism and allosteric modulation properties at the over other Cys-loop receptors. 11 human α1β2γ2 GABAA receptor, the most abundant GABAA Based on the selective PAM profile of (±)-2, we decided to 12 subtype in the adult brain, expressed in Xenopus laevis oocytes further characterize this ligand. We set out to determine if via two-electrode voltage clamp electrophysiology. 13 both enantiomers are active at the α1β2γ2 GABAA receptor. 14 Additionally, we performed mutagenesis experiments to Enantiomer-specific effects would imply a specific drug- 15 identify the binding site of these ligands. receptor interaction, rather than some more generic effect 16 RESULTS AND DISCUSSION The synthesis of the such as altering membrane properties. To assess functional 17 pyrroloindoline framework commenced with protection of effects, we used a similar two-electrode voltage clamp 18 tryptamine (3) to provide carbamate 4 (Scheme 1). Pd- protocol to one previously described by Marotta et al.28 Briefly, 19 catalyzed C2 arylation with iodobenzene under microwave the current responses of three identical EC50 doses of GABA 20 conditions gave 2-phenyl tryptamine 5 in 77% yield.29 Various were recorded, followed by a dose of the test-ligand at 40 μM. 21 approaches were investigated for effecting oxidative After a 30 s incubation, a dose was applied containing both 30 22 cyclization of 5. Although there are many examples of related GABA at its EC50 and the test-ligand at 40 μM. Finally, two 24,31–35 23 cyclizations of tryptamine and tryptophan derivatives, doses of GABA EC50 were applied. The first three GABA doses 24 we found that many of these conditions were unsuitable for establish a baseline of the GABA response at that 25 tryptamine 5, presumably due to the phenyl substituent at C2. concentration, and the purpose of the last two GABA doses is 26 After extensive experimentation, it was found that oxidative to verify proper functioning of the receptor post modulation cyclization of 5 by treatment with N-chlorosuccinimide and control for independent rise in current amplitude. Of the 27 followed by water afforded C3a-hydroxy pyrroloindoline (±)-6 two (±)-2 enantiomers, only (+)-2 showed a meaningful 28 in 85% yield. Reduction of carbamate (±)-6 with Red-Al potentiation of the EC GABA dose, with a mean of 16 ± 4.1%, 29 50 provided the N1-methyl pyrroloindoline (±)-2.31 Attempts to as shown in Figure 2B. 30 render the cyclization of 5 to 6 enantioselective have thus far To determine activity at the α1β2 subtype and consequent 31 35 been unsuccessful; however, the enantiomers of both involvement of the γ2 subunit in potentiation, we performed 32 compounds (±)-6 and (±)-2 can be resolved using preparative 33 the same experiment for this subtype. For (±)-2 a mean SFC with a chiral stationary phase. X-ray crystallography potentiation of 28 ± 5.2% was observed (Figure 2). Similar to 34 confirmed the structure and absolute stereochemistry of (+)-2. the observations for the α1β2γ2 subtype, (+)-2 showed 35 increased potentiation over (–)-2 with mean values of 17 ± 36 Scheme 1. Synthesis of the pyrroloindoline scaffold. 2.6% and 9.2 ± 1.1% respectively (Figure 2B and Table S2). 37 1. CbzCl, i-Pr2NEt These results demonstrate that the γ2 subunit is not required 38 NH2 NHCbz CHCl3 for potentiation of the α1β2γ2 receptor by (±)-2. 39 Pd(OAc) , PhI N 2. MeI, KOH N 2 40 H acetone 2-NO2BzOH Me AgBF , DMF 41 tryptamine 4 86% yield 4 150 ºC, mwave 42 (3) 2 steps 77% yield 43 44 OH NCS, 3 Å MS NHCbz MeCN; 45 2 Ph 46 N N then H2O N Ph 47 Me Cbz 85% yield Me 48 (±)-6 5 49 OH 50 Red-Al 51 PhMe, 80 °C N N 52 97% yield Me Ph Me 53 (±)-2 (+)-2 54 obtained by chiral 55 resolution 56 57 58 59 60 ACS Paragon Plus Environment Page 3 of 9 ACS Medicinal Chemistry Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Figure 2. Functional effects of pyrroloindoline (±)-2 on the α1β2γ2 32 and α1β2 GABAA receptor subtypes. A) Wave forms of the α1β2γ2 33 current responses from a GABA EC50 only dose, 40 μM (±)-2 only Figure 3. Functional characterization of pyrroloindoline (±)-2 at dose, and co-application of GABA EC and 40 μM (±)-2. B) Relative 34 50 the α1β2γ2 GABAA receptor. A) The (±)-2-induced shift in GABA modulation of a GABA EC response of the α1β2γ2 and α1β2 35 50 EC50. A 40 μM concentration of (±)-2 was used here. B) (+)-2 EC50 GABA receptor subtypes by (±)-2 and the individual enantiomers. 36 A co-applied with GABA EC5 doses. The peak current at GABA EC5 *p <0.05; ***p <0.001 (one-way ANOVA). was subtracted from all responses. 37 38 The amplitude of potentiation is dependent on several Having established that pyrroloindoline (+)-2 acts as a PAM 39 factors, among which are both the PAM concentration and the on the α1β2γ2 GABAA receptor, further potentiation 40 GABA concentration at which we tested the modulation. Next, experiments used the GABA EC10-15 instead of EC50. Using the 41 we determined the effect of 40 μΜ (±)-2 on the GABA EC50 EC10-15 allows for a larger potentiation window than EC50, which 42 (ΔEC50((±)-2)) at the α1β2γ2 receptor. The observed (±)-2- enables the detection of more subtle functional differences 43 induced shift in GABA EC50 is 13 μM as shown in Figure 3A and between GABAA mutants or pyrroloindoline analogues. Figures 44 Table S3. This shift is comparable to the induced shift seen for 2B and 4C illustrate this difference in modulation potency for 36 45 this subtype by the benzodiazepine Triazolam, 16-50 μΜ. (±)-2. For the α1β2γ2 subtype, (±)-2 causes a 62% potentiation Moreover, we wanted to determine the potency of the pure 46 of the GABA EC10 response, while at the GABA EC50 this is only enantiomer (+)-2. Well-studied modulators, such as 47 17%. flurazepam and zolpidem, have EC50s in the nanomolar range 48 GABA activates the GABAA receptor through binding in the when co-applied with GABA EC , being 270 nM and 340 nM, 49 2-5 ECD at the interface of the β+/α- subunits. Besides this respectively.37 The PAM tested here, (+)-2, appears to be less 50 orthosteric site, several allosteric binding sites have been potent with an EC50 of 110 μΜ when co-applied with GABA EC5, established, of which the benzodiazepine site (BZ) in the ECD 51 as shown in Figure 3B and Table S3. 52 at the α+/γ- interface is the most well-known.38 More recently, 53 a distinct binding site in the ECD at the α+/β- interface has 39,40 54 been identified for the ligand CGS9895. In addition to 55 binding sites in the ECD, several anesthetics and neurosteroids 56 affect channel activity through binding in the TMD. Recent X- 57 58 59 60 ACS Paragon Plus Environment ACS Medicinal Chemistry Letters Page 4 of 9

ray crystal structures and cryo-EM structures have shed light α1(S297I)β2(N289I)γ2(S319I) to probe for anesthetic sites in 1 on the TM residues involved in binding.41,42 the TMD.40,44 All three ECD mutants were potentiated to a 2 similar extent as the WT receptor (mean 62 ± 6.1%). However, 3 the triple TMD mutant was not affected by (±)-2 (mean 1.0 ± 4 3.0%) as shown in Figure 4C and Table S4. These results 5 indicate that (±)-2 does not assert its potentiating affects 6 through binding at the interfaces in the ECD, but on one or 7 more interfaces in the TMD. 8 To determine which specific interfaces are involved in 9 binding in the TMD, we performed potentiation experiments 10 for the single and double mutants of α1β2γ2, as well as the 11 α1β2 subtype (Figure 4B). The mean potentiation in both 12 mutants with single mutations in the α1 and γ2 subunits 13 resembles that of the α1β2γ2 WT receptor. Only the single and 14 double mutant receptors that contain a mutation in the β2 15 subunit demonstrate greatly reduced potentiation, suggesting 16 involvement of the β2 subunit in binding. For the α1β2 17 subtype, greater variability among the mutants has been 18 observed, but the same general trend appears. The β2(N289I) 19 mutation is located in the TMD, close to the top of TM2, at the 20 β+/α- interface (Figure 4A). This residue has been implicated in 21 the binding of several anesthetics, such as etomidate, 40,45 22 propofol, and loreclezole. 23 We performed potentiation experiments on these GABAA 24 mutants similarly as described in Figure 2, however here we 25 co-applied the 40 μM PAM with an EC10-15 dose of GABA. To 26 verify the EC10-15 values of the constructed mutant receptors, 27 we determined the full dose-response relationships and found 40,44 28 EC50 values similar those reported previously. (Table S5). 29 Having characterized lead compound (+)-2, we aimed to 30 optimize the potency of this ligand family by way of exploring 31 substitutions at N1, C3a, C5, and C8a. Derivatization of the 32 Figure 4. Functional effects of pyrroloindoline (±)-2 on α1β2γ2 and pyrroloindoline scaffold was undertaken with enantioenriched 33 α1β2 GABAA receptor mutants. A) Side view of the human α1β2γ2 compounds 2 and 6 (Scheme 2). For each of the analogues 34 GABAA receptor with the probed residues highlighted in pink (PDB synthesized, both enantiomers were prepared for evaluation 35 ID: 6D6T). B) Extracellular view into the pore with GABA and BZ of their ability to modulate GABAA receptors; however, for 36 sites indicated with arrows. C) Relative modulation of GABA10-15 simplicity, the chemistry is depicted on the (+) enantiomers of 37 responses by (±)-2. ECD mutants and TMD mutants of α1β2γ2 are 2 and 6 in Scheme 2. From carbamate (+)-6, Pd-catalyzed in blue and dark red respectively. TMD mutants of α1β2 are in light 38 deprotection of the Cbz group afforded the N1–H red. **p <0.01; ****p <0.0001 (one-way ANOVA). 39 pyrroloindoline (+)-7. Derivatization of C3a was examined to 40 In order to determine the binding site for the PAM (±)-2, we interrogate the possibility of the C3a hydroxyl group acting as 41 performed mutagenesis on residues that have been implicated a hydrogen bond donor. Deoxyfluorination of (+)-6 using diethylaminosulfur trifluoride followed by carbamate 42 in binding of known modulators. For the first screen we 38 40,43 reduction with Red-Al furnished tertiary fluoride (+)-9. 43 selected α1(H129R) and α1(Y209Q) to probe the BZ-site, 39 Methylation of the C3a hydroxy group of (+)-2 under standard 44 β2(Q88C) to probe the α+/β- site, and triple mutant conditions gave C3a-methoxy pyrroloindoline (+)-10. 45 46 Scheme 2. Derivatization of the pyrroloindoline framework. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 5 of 9 ACS Medicinal Chemistry Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Recognizing that physostigmine (1) possesses oxidation at 26 C5, we also sought to derivatize this position of the 27 pyrroloindoline framework with both electron-donating and 28 electron-withdrawing substituents. Electrophilic aromatic 29 substitution of (+)-2 with N-iodosuccinimide afforded aryl 30 iodide (–)-11 (Scheme 2). Cu-catalyzed trifluoromethylation 31 of the iodoarene gave (+)-12.46 Bromination was also feasible 32 using N-bromosuccinimide to furnish (+)-13. From the aryl 33 bromide, Cu-catalyzed methoxylation provided methoxy 34 analogue (+)-14.47 Additionally, Buchwald-Hartwig coupling 35 of aryl bromide (+)-13 gave morpholine (–)-15.48 36 We also sought to introduce structural variations on the 37 C8a aryl group. Using 1-bromo-4-iodobenzene, Pd-catalyzed 38 C2 arylation of protected tryptamine 4 gave an aryl bromide 39 analogue that was advanced to pyrroloindoline (±)-16 (see 40 Scheme S1 for synthetic details). 41 Functional evaluation of the series of pyrroloindoline 42 derivatives was conducted using two-electrode voltage 43 clamp electrophysiology as described earlier for (±)-2. 44 General trends will be discussed first. Most of the derivatives 45 do not demonstrate agonist behaviors, except for 46 compounds (–)-11 and (+)-13, which only activated the 47 receptor with very low efficacy (Figure 6A). Generally, all 48 derivatives demonstrate a similar activity pattern for the two 49 enantiomers as we have observed for (±)-2, with only the S,S- 50 enantiomer demonstrating activity. One exception to this is 51 the aryl bromide 16, for which both enantiomers show 52 substantial potentiation. It is also worth noting that 53 morpholines (+)-15 and (–)-15, and aryl iodide (+)-11 and (–)- Figure 5. Functional effects of pyrroloindoline derivatives at the 54 11, have reversed signs for their optical rotations as α1β2γ2 GABAA receptor. A) N1, C2, C3-substituted derivatives. 55 compared to all other derivatives. B) C5-substituted derivatives. *p <0.05; **p <0.01; ***p <0.001; 56 ****p <0.0001 (one-way ANOVA). 57 58 59 60 ACS Paragon Plus Environment ACS Medicinal Chemistry Letters Page 6 of 9

Changes at N1 resulted in decreased potentiation relative 1 to the enantiomerically pure parent ligand (+)-2 (125 ± 15%), 2 with the N1-protio compound (+)-7 and the N1-Cbz 3 compound (+)-6 giving potentiation values of 41 ± 3.0% and 4 9.6 ± 5.2%, respectively (Figure 5A and Table S6). Substitution 5 of the C3a hydroxyl with fluorine ((+)-9) gives similar 6 potentiation (100 ± 11%) to (+)-2, whereas methylation of 7 the hydroxyl group ((+)-10) results in a substantial reduction 8 in activity (21 ± 3.0%). 9 Next, we looked at the C5-substituted derivatives (Figure 10 5B and Table S6). The presence of a methoxy ((+)-14) or 11 morpholino ((+)-15) substituent at C5 drastically reduced the 12 potentiation efficacy to 17 ± 3.2% and –4.8 ± 1.8% 13 respectively. Potentiation by trifluoromethyl compound (+)- 14 12 resembled that of (+)-2 at 108 ± 9.5%. Surprisingly, 15 introduction of a halogen (Br or I) at C5 greatly increased 16 potentiation with a modulation of 213 ± 21% for (+)-13 and 17 231 ± 24% for (–)-11 respectively (Figure 5B and 6A). This 18 structure-activity relationship could indicate the presence of 19 a halogen bonding binding interaction. These two ligands 20 appear to have the largest potentiation effects on the 21 α1β2γ2 GABAA receptor at 40 μΜ of all the derivatives 22 evaluated here. Therefore, we attempted to determine a full 23 dose-response relationship for these two PAMs, however 24 solubility problems at concentrations greater than 100 μΜ 25 prevented this. (Figure S2) Additionally, we determined the 26 GABA ΔEC50 shift due to 40 μΜ (–)-11 or (+)-13 at the α1β2γ2 receptor; we observed a 9 μM and 16 μM shift for (–)-11- and 27 (+)-13 respectively (Figure 6B and Table S2). These values are 28 similar to that observed for (±)-2. 29 30 31 32 Figure 6. Functional characterization of pyrroloindoline (–)-11 33 and (+)-13 at the α1β2γ2 GABAA receptor. A) Wave forms of the α1β2γ2 current responses from a GABA EC only dose, 40 μM 34 10 (±)-11 or (+)-13 only dose, and co-application of GABA EC and 35 10 40 μM PAM. B) PAM-induced shift in GABA EC50. A 40 μM 36 concentration of (±)-11 and (+)-13 was used here. 37 38 Comparing the different effects of C5 substitution and 39 structural variations at the C8a aryl group, recall that 40 methoxy analogue (+)-14 did not exhibit any PAM properties, 41 nor did methoxyarene (±)-SI-5 (Table S5). However, bromide (+)-13 demonstrated increased PAM properties relative to 42 (+)-2. Considering the spatial positioning of the bromine at 43 C5, we asked whether a ligand with a 4-Br-Ph at C8a would 44 also possess PAM properties. Indeed, both aryl bromides (+)- 45 16 (98 ± 8.3%) and (–)-16 (155 ± 11%) showed comparable 46 potency to (+)-2 (Figure 5B and Table S6). It is surprising that 47 both enantiomers of 16 are active, and we hypothesize that 48 the R,R-enantiomer (–)-16 might be able to bind in an “upside 49 down” orientation, in which the bromine occupies the same 50 position as the bromine of ligand (+)-13. Figure 7 depicts an 51 overlay of the two chemical structures, (+)-13 and (–)-16, to 52 illustrate this. (–)-16 is shown at a slightly rotated orientation 53 (looking down C3a and C8a instead of C2) to mimic the 54 orientation of (+)-13, which indeed resembles this structure. 55 These results indicate that not only is the para-position of the 56 C8a-phenyl substituent permissive to halide substitution, but 57 it is possible that (–)-16, the R,R-enantiomer, is able to fit the 58 59 60 ACS Paragon Plus Environment Page 7 of 9 ACS Medicinal Chemistry Letters

binding pocket in the upside down orientation, unlike the We thank Alex Maolanon and Katie Chan for early synthesis 1 other ligands tested in this study. efforts, as well as Dr. Chris B. Marotta and Dr. Kristina Daeffler 2 for performing the preliminary Cys-loop screen. We are grateful 3 to Dr. Scott Virgil and the Caltech Center for Catalysis and 4 Chemical Synthesis for access to analytical equipment and 5 assistance with performing preparative chiral HPLC and SFC 6 resolutions. S. E. R. is a Heritage Medical Research Institute 7 Investigator. Financial support from the NIH (S. E. R. R35GM118191-01) is gratefully acknowledged. 8 9 ABBREVIATIONS 10 GABAA, γ-aminobutyric acid type A; PAM, positive allosteric 11 modulator; pLGIC, pentameric ligand-gated ion channel; BZ, 12 benzodiazepine; SEM, standard error of the mean. 13 14 REFERENCES 15 (1) Rudolph, U.; Möhler, H. GABAA Receptor Subtypes: 16 Therapeutic Potential in Down Syndrome, Affective Disorders, 17 Schizophrenia, and Autism. 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For Table of Contents Use Only: 1 2 Synthesis and Biological Evaluation of Pyrroloindolines as Positive Allosteric Modulators of the 3 α1β2γ2 GABA Receptor 4 A 5 Annet E. M. Blom, Justin Y. Su, Lindsay M. Repka, Sarah E. Reisman*, Dennis A. Dougherty* 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 9 60 ACS Paragon Plus Environment